Gene activation is a highly regulated process that requires the coordinated action of proteins to relieve chromatin repression and to promote transcriptional activation. Nuclear histone acetyltransferase (HAT) enzymes provide a mechanistic link between chromatin destabilization and gene activation by acetylating the a nitrogens of specific lysine residues within the N-terminal tails of core histones to facilitate DNA access by transcriptional activators. Several different HAT enzymes have been identified including GCN5 [from Tetrahymena (tGCN5), yeast (yGCN5), human, Drosophilia and Arabadopsis], yeast ESA1 (yESA1), and human PCAF (hPCAF), CBP/p300, TAFII250, Tip60, ACTR, and SRC-1. Functional characterization of a subset of these enzymes have revealed that they have overlapping, yet distinct histone substrate specificities. Moreover, a subset of the HAT proteins, including hPCAF and CBP/p300, have been found to specifically acetylate non-histone/transcription factor substrates such as the p53 tumor suppressor. The overall objective of this proposal is to use the tGCN5, hPCAF and yESA1 proteins as a model to elucidate the substrate specificity and catalytic mechanism of HAT enzymes. We will use a combined approach of high resolution structure determinations with in vitro substrate binding and catalysis measurements. Towards this goal, we have recently determined the high resolution X-ray crystal structure of yGCN5, and have compared it to related acetyltransferases. This comparison leads us to hypothesize that HAT enzymes contain a structurally conserved core for substrate acetylation, and that residues within and directly and C-terminal to this core are responsible for substrate specific binding and catalysis. In this grant application, we propose to test our hypothesis and to extend our studies with the following experiments, (1) Determine the structure of tGCN5 alone, in binary complex with acetyl-CoA (A-CoA) cofactor and in ternary complex with CoA/histone H3 substrate; (2) Determine the structure of hPCAF in binary complex with A-CoA and in ternary complexes with CoA/histone H3 and CoA/p53 peptide substrates; (3) Determine the structure of yESA1 in binary complex with CoA and in ternary complex with CoA/histone H4 substrate; (4) Measure the equilibrium and kinetic parameters for substrate binding and acetylation using wild-type and mutant peptide substrates and HAT proteins. These studies will provide a paradigm for the structure and function of HAT enzymes, and will provide a scaffold for the design of small molecule compounds to inhibit specific HAT enzymes for application in basic research and medicine.